Remodeling Isoprene Pyrophosphate Metabolism for Promoting Terpenoids Bioproduction

Terpenoids are the largest family of natural products.They are made from the building block isoprene pyrophosphate(IPP),and their bioproduction using engineered cell factories has received a great deal of attention.To date,the insufficient metabolic supply of IPP remains a great challenge for the efficient synthesis of terpenoids.In this work,we discover that the imbalanced metabolic flux distribution between the central metabolism and the IPP supply hinders IPP accumulation in Bacillus subtilis(B.subtilis).Therefore,we remodel the IPP metabolism using a series of genetically encoded two-input-multioutput(TIMO)circuits that are responsive to pyruvate or/and malonyl-CoA,resulting in an IPP pool that is significantly increased by up to four-fold.As a proof-of-concept validation,we design an IPP metabolism remodeling strategy to improve the production of three valuable terpenoids,including menaquinone-7(MK-7,4.1-fold),lycopene(9-fold),andβ-carotene(0.9-fold).In particular,the titer of MK-7 in a 50-L bioreactor reached 1549.6 mg·L^(-1),representing the highest titer reported so far.Thus,we propose a TIMO genetic circuits-assisted IPP metabolism remodeling framework that can be generally used for the synergistic fine-tuning of complicated metabolic modules to achieve the efficient bioproduction of terpenoids.

Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening

Alpha-lactalbumin(α-LA)is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants.In this study,Bacillus subtilis RIK1285 harboring AprE signal peptide(SP)was selected as the original strain for the production ofα-LA.It was found thatα-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation supernatant.The original strain most likely only producedα-LA intracellular,but not extracellular.To improve the expression and secretion ofα-LA in RIK1285,a library of 173 homologous SPs from the B.subtilis 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in RIK1285.SP YjcN was determined to be the best signal peptide.Bands in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal peptide.In addition,different promoters(P_(aprE),P_(43),and P_(glv))were compared and applied.The results indicated that the strain RIK1285-pBE-P_(glv)-YjcN-LALBA had the highestα-LA yield,reaching 122.04μg/mL.This study demonstrates successful expression and secretion of humanα-LA in B.subtilis and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.

Food synthetic biology-driven protein supply transition: From animal-derived production to microbial fermentation

Animal-derived protein production is one of the major traditional protein supply methods,which continues to face increasing challenges to satisfy global needs due to population growth,augmented individual protein consumption,and aggravated environmental pollution.Thus,ensuring a sustainable protein source is a considerable challenge.The emergence and development of food synthetic biology has enabled the establishment of cell factories that effectively synthesize proteins,which is an important way to solve the protein supply problem.This review aims to discuss the existing problems of traditional protein supply and to elucidate the feasibility of synthetic biology in the process of protein synthesis.Moreover,using artificial bioengineered milk and artificial bioengineered eggs as examples,the progress of food protein supply transition based on synthetic biology has been systematically summarized.Additionally,the future of food synthetic biology as a potential source of protein has been also discussed.By strengthening and innovating the application of food synthetic biology technologies,including genetic engineering and high-throughput screening methods,the current limitations of artificial foods for protein synthesis and production should be addressed.Therefore,the development and industrial production of new food resources should be explored to ensure safe,high-quality,and sustainable global protein supply.

A review on current conventional and biotechnical approaches to enhance biosynthesis of steviol glycosides in Stevia rebaudiana

Stevia rebaudiana Bertoni is commonly called stevia and mostly found in the north east regions of South America.It is an herbaceous and shrubby plant belonging to the Asteraceae family.Stevia is considered as a natural sweetener and a commercially important plant worldwide.The leaves of S.rebaudiana contain steviol glycosides(SGs)which are highly potent and non-caloric sweeteners.The sweetening property of S.rebaudiana is contributed to the presence of these high potency,calorie free steviol glycosides.SGs are considerably suitable for replacing sucrose and other artificial sweetening agents which are used in different industries and pharmaceuticals.SGs amount in the plant mostly varies from 8%to 10%,and the enhancement of SGs is always in demand.These glycosides have the potential to become healthier alternatives to other table sugars for having desirable taste and zero calories.SGs are almost 300 times sweeter than sucrose.Being used as alternative sugar intensifier the commercial value of this plant in biopharmaceutical,food and beverages industries and in international market is increasing day by day.SGs have made stevia an important part of the medicinal world as well as the food and beverage industry,but the limited production of plant material is not fulfilling the higher global market demand.Therefore,researchers are working worldwide to increase the production of important SGs through the intercession of different biotechnological approaches in S.rebaudiana.This review aims to describe the emerging biotechnological strategies and approaches to understand,stimulate and enhance biosynthesis of secondary metabolites in stevia.Conventional and biotechnological methods for the production of steviol glycosides have been briefly reviewed and discussed.

Current progress and prospects of enzyme technologies in future foods

Enzyme technologies are widely used in the food industry due to their advantages of high efficiency,specificity,and safety.Recently,“future foods”is emerging as a new research hotspot with healthier foods that are more nutritious,delicious,and sustainable;however,these foods still have problems with texture,nutrition,and flavor.Advances in enzyme technology have enabled the development of new tools and approaches to better manipulate food textures and nutritional aspects.In this review,we summarize enzyme technology applications in future food production,focusing on food texture,safety,and flavors.Furthermore,we discuss the prospects of enzyme-based technologies for future food production,including the modification of enzyme activities,the development of suitable food-grade hosts for enzyme production,and the optimiza-tion synergistic multi-enzyme systems.

Yeast surface display of leech hyaluronidase for the industrial production of hyaluronic acid oligosaccharides

Leech hyaluronidase(LHyal)is a hyperactive hyaluronic acid(HA)hydrolase that belongs to the hyaluronoglu-curonidase family.Traditionally,LHyal is extracted from the heads of leeches,but the recent development of the Pichia pastoris recombinant LHyal expression method permitted the industrial production of size-specific HA oligosaccharides.However,at present LHyal expressed by recombinant yeast strains requires laborious protein purification steps.Moreover,the enzyme is deactivated and removed after single use.To solve this problem,we developed a recyclable LHyal biocatalyst using a yeast surface display(YSD)system.After screening and charac-terization,we found that the cell wall protein Sed1p displayed stronger anchoring to the P.pastoris cell wall than other cell wall proteins.By optimizing the type and length of the linkers between LHyal and Sed1p,we increased the activity of enzymes displayed on the P.pastoris cell wall by 50.34%in flask cultures.LHyal-(GGGS)6-Sed1p activity further increased to 3.58×105 U mL−1 in fed-batch cultivation in a 5 L bioreactor.Enzymatic prop-erty analysis results revealed that the displayed LHyal-(GGGS)6-Sed1p generated the same oligosaccharides but exhibited higher thermal stability than free LHyal enzyme.Moreover,displayed LHyal-(GGGS)6-Sed1p could be recovered easily from HA hydrolysis solutions via low-speed centrifugation and could be reused at least 5 times.YSD of LHyal not only increased the utilization efficiency of the enzyme but also simplified the purification pro-cess for HA oligosaccharides.Thus,this study provides an alternative approach for the industrial preparation of LHyal and HA oligosaccharides.

Engineering Escherichia coli for high-yield production of ectoine

Ectoine is a natural macromolecule protector and synthesized by some extremophiles.It provides protections against radiation-mediated oxidative damages and is widely used as a bioactive ingredient in pharmaceutics and cosmetics.To meet its growing commercial demands,we engineered Escherichia coli strains for the high-yield production of ectoine.The ectABC gene cluster from the native ectoine producer Halomonas elongata was intro-duced into different Escherichia coli(E.Coil)strains via plasmids and 0.8 g L^(-1)of ectoine was produced in flask cultures by engineered E.coli BL21(DE3).Subsequently,we designed the ribosome-binding sites of the gene cluster to fine-tune the expressions of genes ectA,ectB,and ectC,which increased the ectoine yield to 1.6 g L^(-1).After further combinatorial overexpression of Corynebacterium glutamicum aspartate kinase mutant(G1A,C932T)and the H.elongate aspartate-semialdehyde dehydrogenase to increase the supply of the precursor,the titer of ectoine reached to 5.5 g L^(-1)in flask cultures.Finally,the engineered strain produced 60.7 g L^(-1)ectoine in fed-batch cultures with a conversion rate of 0.25 g/g glucose.

Synergetic engineering of Escherichia coli for efficient production of L-tyrosine

L-Tyrosine,an aromatic non-essential amino acid,is the raw material for many important chemical products,including levodopa,resveratrol,and hydroxytyrosol.It is widely used in the food,drug,and chemical industries.There are many studies on the synthesis of L-tyrosine by microorganisms,however,the low titer of L-tyrosine limited the industrial large-scale production.In order to enhance L-tyrosine production in Escherichia coli,the expression of key enzymes in the shikimate pathway was up-or down-regulated.The L-tyrosine transport system and the acetic acid biosynthesis pathway were modified to further enhance L-tyrosine production.In addition,the phosphoketolase pathway was introduced in combination with cofactor engineering to redirect carbon flux to the shikimate pathway.Finally,after adaptive laboratory evolution to low pH an optimal strain was obtained.The strain can produce 92.5 g/L of L-tyrosine in a 5-L fermenter in 62 h,with a yield of 0.266 g/g glucose.

Microbial chassis design and engineering for production of amino acids used in food industry

Rational microbial chassis design and engineering for improving production of amino acids have attracted a considerable attention.l-glutamate,l-lysine,l-threonine and l-tryptophan are the main amino acids demanded in the food industry.Systems metabolic engineering and synthetic biology engineering generally are believed as the comprehensive engineering approaches to obtain rationally designed strains and construct high-performance platforms for amino acids.The strate-gies focus on microbial chassis characterization optimization,precise metabolic engineering such as promoter engineer-ing,modular pathway engineering,transporter engineering,and dynamic switch systems application,and global genome streamline engineering to reduce cell burden.In this review,we summarized the efficient engineering strategies to optimize Corynebacterium glutamicum and Escherichia coli cell factories for improving the production of l-glutamate,l-lysine,l-threonine,and l-tryptophan.

Characterization of putative mannoprotein in Kluyveromyces lactis for lactase production

Lactase is a member of theβ-galactosidase family of enzymes that can hydrolyze lactose into galactose and glucose.However,extracellular lactase production was still restricted to the process of cell lysis.In this study,lactase-producing Kluyveromyces lactis JNXR-2101 was obtained using a rapid and sensitive method based on the fluorescent substrate 4-methylumbelliferyl-β-D-galactopyranoside.The purified enzyme was identified as a neutral lactase with an optimum pH of 9.To facilitate extracellular production of lactase,a putative mannoprotein KLLA0_E01057g of K.lactis was knocked out.It could effectively promote cell wall degradation and lactase production after lyticase treatment,which showed potential on other extracellular enzyme preparation.After optimizing the fermentation conditions,the lactase yield from mannoprotein-deficient K.lactis JNXR-2101ΔE01057g reached 159.62 U/mL in a 5-L fed-batch bioreactor.

Design of artificial small regulatory trans-RNA for gene knockdown in Bacillus subtilis

Bacillus subtilis as the Gram-positive model bacterium has been widely used in synthetic biology and biotechnology while the regulatory RNA tools for B.subtilis are still not fully explored.Here,a bottom-up approach is proposed for designing artificial trans-acting sRNAs.By engineering the intrinsic sRNA SR6,a minimized core scaffold structure consisting of an 8 bp stem,a 4 nt loop,and a 9 nt polyU tail was generated and proven to be sufficient for constructing sRNAs with strong repression activity(83%).Moreover,we demonstrate this artificial sRNA system functions well in an hfq-independent manner and also achieves strong repression efficiency in Escherichia coli(above 80%).A structure-based sRNA design principle was further developed for the automatic generation of custom sRNAs with this core scaffold but various sequences,which facilitates the manipulation and avoids structure disruption when fusing any base-pairing sequence.By applying these auto-designed sRNAs,we rapidly modified the cell morphology and biofilm formation,and regulated metabolic flux toward acetoin biosynthesis.This sRNA system with cross-species regulatory activities not only enriched the gene regulation toolkit in synthetic biology for B.subtilis and E.coli but also enhanced our understanding of trans-acting sRNAs.

Engineering Saccharomyces cerevisiae for enhanced(–)-α-bisabolol production

(–)-α-Bisabolol is naturally occurring in many plants and has great potential in health products and pharma-ceuticals.However,the current extraction method from natural plants is unsustainable and cannot fulfil the increasing requirement.This study aimed to develop a sustainable strategy to enhance the biosynthesis of(–)-α-bisabolol by metabolic engineering.By introducing the heterologous gene MrBBS and weakening the competitive pathway gene ERG9,a de novo(–)-α-bisabolol biosynthesis strain was constructed that could produce 221.96 mg/L(–)-α-bisabolol.Two key genes for(–)-α-bisabolol biosynthesis,ERG20 and MrBBS,were fused by a flexible linker(GGGS)3 under the GAL7 promoter control,and the titer was increased by 2.9-fold.Optimization of the mevalonic acid pathway and multi-copy integration further increased(–)-α-bisabolol production.To promote product efflux,overexpression of PDR15 led to an increase in extracellular production.Combined with the optimal strategy,(–)-α-bisabolol production in a 5 L bioreactor reached 7.02 g/L,which is the highest titer reported in yeast to date.This work provides a reference for the efficient production of(–)-α-bisabolol in yeast.

An effective cytokine combination for ex vivo expansion of porcine muscle stem cells

Cultured meat technology is a novel and promising strategy for sustainable and effective meat production.Muscle stem cells are widely used as seed cell populations because of their ease of access and myogenic differentiation potential.However,muscle stem cells are difficult to continuously propagate ex vivo and are heavily dependent upon serum for survival and growth,which impedes their commercial use as a cultured meat source.Herein,we identified an effective four-cytokine combination to promote long-term proliferation of porcine muscle stem cells using a serial elimination screening approach,which was consisted of long chain human insulin growth factor-1,platelet derived growth factor BB,basic fibroblast growth factor,and epidermal growth factor.The expansion of muscle stem cells with the four-cytokine combination achieved a 6.31×107-fold increase in cell number after 28 days of culture with retained cell myogenic differentiation potential,and most importantly,reduced the amount of fetal bovine serum required for cell culture by at least 5%.Furthermore,the four-cytokine combination exerted the pro-proliferative function by activating PI3K/Akt/mTOR and MEK/ERK signaling pathways.This approach provides for a new means by which to industrialize the production of cultured meat.

Metabolic engineering of Escherichia coli for the production of Lacto-N-neotetraose(LNnT)

Lacto-N-neotetraose(LNnT),one of the most important human milk oligosaccharides,can be used as infants’food addi-tives.Nowadays,extraction,chemical and biological synthesis were utilized to obtain LNnT,while these methods still face some problems such as low yield and high cost.The aim of current work is to construct a de novo biosynthesis pathway of LNnT in E.coli K12 MG1655.The lgtA and lgtB were first expressed by a plasmid,resulting in a LNnT titer of 0.04 g/L.To improve the yield of LNnT on substrate lactose,lacZ and lacI were knocked out,and lacY was over-expressed.As a result,the yield of LNnT on lactose increased from 0.01 to 0.09 mol/mol,and the titer of LNnT elevated to 0.41 g/L.In addition,the pathway was regulated using the titer of Lacto-N-triose II(LNTII)as a measure,and obtained a high titer strain of LNnT for 1.04 g/L.Finally,the gene expressions were fine-tuned,the titer of LNnT reached 1.2 g/L,which was 93%higher than the control strain,and the yield on lactose reached 0.28 mol/mol.The engineering strategy of pathway construction and modulation used in this study is applicable to facilitate the microbial production of other metabolites in E.coli.

Elimination of ethyl carbamate in fermented foods

Ethyl carbamate (EC) is a common harmful by-product in fermented foods and can be derived from raw materials or formed during the fermentation or even in the post-treatment.Its content can be affected by temperature,microorganisms,technological processes,and other factors.In this review,we summarized the technologies and their applications for EC elimination in fermented foods in the light of upstream processes,such as raw material pretreatment,lower temperature,breeding microorganisms,and exogenous substances,to the downstream enzymatic method and physical adsorption.The review covers different strategies to eliminate EC,such as strain breeding,genetic engineering,enzyme,and physicochemical strategies.By comparing these strategies,practical strategies for the efficient elimination of EC in different fermented foods on an industrial scale have been discussed and proposed in consideration of flavor,food safety and feasibility of an industrial application.

AI-assisted food enzymes design and engineering:a critical review

Food enzymes are basic components used for food processing.Through catalysis,food enzymes can function as removing allergy,enriching absorbable nutrients,improving food texture,and adjusting flavors.Food enzymes work in various conditions,which brought out the need for engineering these enzymes with harsh environment tolerance and higher catalytic efficiency.Artificial intelligence(AI)has recently provided solutions for structural modeling,finding modification hot spots,and guiding mutations toward specific needs,which greatly benefit enzyme engineering.AI-based tools showed great advantages in cutting down the computational time,enabling higher prediction accuracy,and providing trainable models suited for wide uses.In this review,we describe the functions and uses of food enzymes,as well as their utility limitations.The necessity and advantages of using AI-based tools in enzyme engineering,and the differences between using traditional and AI-based tools are mainly discussed.Few AI-based tools for enzyme engineering were introduced and described their function.The perspective of using AI tools and the future challenges were discussed.

Gene knockdown by structure defined single-stem loop small non-coding RNAs with programmable regulatory activities

Gene regulation by trans-acting small RNAs(sRNAs)has considerable advantages over other gene regulation strategies.However,synthetic sRNAs mainly take natural sRNAs(MicC or SgrS)as backbones and comprise three functional elements folding into two or more stem-loop structures:an mRNA base pairing region,an Hfq-binding structure,and a rho-independent terminator.Due to limited numbers of natural sRNAs and complicated backbone structures,synthetic sRNAs suffer from low activity programmability and poor structural modularity.Moreover,natural sRNA backbone sequences may increase the possibility of unwanted recombination.Here,we present a bottom-up approach for creating structure defined single-stem loop small non-coding RNAs(ssl-sRNAs),which contain a standardized scaffold of a 7 bp-stem-4 nt-loop-polyU-tail and a 24 nt basing pairing region covering the first eight codons.Particularly,ssl-sRNA requires no independent Hfq-binding structure,as the polyU tail fulfills the roles of binding Hfq.A thermodynamic-based scoring model and a web server sslRNAD(http://www.kangzlab.cn/)were developed for automated design of ssl-sRNAs with well-defined structures and programmable activities.ssl-sRNAs displayed weak polar effects when regulating polycistronic mRNAs.The ssl-sRNA designed by sslRNAD showed regulatory activities in both Escherichia coli and Bacillus subtilis.A streamlined workflow was developed for the construction of customized ssl-sRNA and ssl-sRNA libraries.As examples,the E.coli cell morphology was easily modified and new target genes of ergothioneine biosynthesis were quickly identified with ssl-sRNAs.ssl-sRNA and its designer sslRNAD enable researchers to rapidly design sRNAs for knocking down target genes.

Enhancement of phycocyanobilin biosynthesis in Escherichia coli by strengthening the supply of precursor and artificially self-assembly complex

Phycocyanobilin(PCB)is widely used in healthcare,food processing,and cosmetics.Escherichia coli is the common engineered bacterium used to produce PCB.However,it still suffers from low production level,pre-cursor deficiency,and low catalytic efficiency.In this study,a highly efficient PCB-producing strain was created.First,chassis strains and enzyme sources were screened,and copy numbers were optimized,affording a PCB titer of 9.1 mg/L.Most importantly,the rate-limiting steps of the PCB biosynthetic pathway were determined,and the supply of precursors necessary for PCB synthesis was increased from endogenous sources,affording a titer of 21.4 mg/L.Then,the key enzymes for PCB synthesis,HO1 and PcyA,were assembled into a multi-enzyme complex using the short peptide tag RIAD-RIDD,and 23.5 mg/L of PCB was obtained.Finally,the basic con-ditions for PCB fermentation were initially determined in 250 mL shake flasks and a 5-L bioreactor to obtain higher titers of PCB.The final titer of PCB reached 147.0 mg/L,which is the highest reported titer of PCB so far.This research provided the foundation for the industrial production of PCB and its derivatives.

Efficient stereoselective hydroxylation of deoxycholic acid by the robust whole-cell cytochrome P450 CYP107D1 biocatalyst

Deoxycholic acid(DCA)has been authorized by the Federal Drug Agency for cosmetic reduction of redundant submental fat.The hydroxylated product(6β-OH DCA)was developed to improve the solubility and pharmaceutic properties of DCA for further applications.Herein,a combinatorial catalytic strategy was applied to construct a powerful Cytochrome P450 biocatalyst(CYP107D1,OleP)to convert DCA to 6β-OH DCA.Firstly,the weak expression of OleP was significantly improved using pRSFDuet-1 plasmid in the E.coli C41(DE3)strain.Next,the supply of heme was enhanced by the moderate overexpression of crucial genes in the heme biosynthetic pathway.In addition,a new biosensor was developed to select the appropriate redox partner.Furthermore,a cost-effective whole-cell catalytic system was constructed,resulting in the highest reported conversion rate of 6β-OH DCA(from 4.8%to 99.1%).The combinatorial catalytic strategies applied in this study provide an efficient method to synthesize high-value-added hydroxylated compounds by P450s.

In silico cell factory design driven by comprehensive genome‑scale metabolic models:development and challenges

Genome-scale metabolic models(GEMs)have been widely used to design cell factories in silico.However,initial flux balance analysis only considers stoichiometry and reaction direction constraints,so it cannot accurately describe the distribution of metabolic flux under the control of various regulatory mechanisms.In the recent years,by introducing enzymology,thermodynamics,and other multiomics-based constraints into GEMs,the metabolic state of cells under different conditions was more accurately simulated and a series of algorithms have been presented for microbial phenotypic analysis.Herein,the development of multiconstrained GEMs was reviewed by taking the constraints of enzyme kinetics,thermodynamics,and transcriptional regulatory mechanisms as examples.This review focused on introducing and summarizing GEMs application tools and cases in cell factory design.The challenges and prospects of GEMs development were also discussed.